A predicted alpha -helix mediates targeting of the proprotein convertase PC1 to the regulated secretory pathway

The proprotein convertase PC1 is a protease whose activity is largely confined to the dense core secretory granules of neuroendocrine cells. Efficient processing of PC1 substrates in granules requires a mechanism that will both limit the activity of the enzyme to these organelles and promote its targeting to the nascent secretory granules. In the current study, we provide evidence that targeting of PC1 to secretory granules is mediated by alpha-helical structures in its C-terminal tail and, at least in part, is dependent on interactions with specific components of the secretory granule membrane.     

Protein targeting to dense-core secretory granules

Regulated secretory proteins are stored within specialized vesicles known as secretory granules. It is not known how proteins are sorted into these organelles. Regulated proteins may possess targeting signals which interact with specific sorting receptors in the lumen of the trans-Golgi network (TGN) prior to their aggregation to form the characteristic dense-core of the granule. Alternatively, sorting may occur as the result of specific aggregation of regulated proteins in the TGN. Aggregates may be directed to secretory granules by interaction of a targeting signal on the surface with a sorting receptor. Novel targeting signals which confer on regulated proteins a tendency to aggregate under certain conditions, and in so doing cause them to be incorporated into secretory granules, have been implicated. Specific targeting signals may also play a role in directing membrane proteins to secretory granules.     

Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

The lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules

Phogrin, a transmembrane glycoprotein of neuroendocrine cells, is localized to dense-core secretory granules. We have investigated the subcellular targeting of phogrin by analyzing the sorting of a series of deletion mutants to the regulated pathway of secretion in AtT20 cells. The lumenal domain as a soluble protein was efficiently routed to granules, based on a combination of morphological analysis and secretion studies. Sorting was not dependent on a candidate targeting signal consisting of an N-terminal conserved cysteine-rich motif. Both the pro-region and the lumenal domain of mature, post-translationally processed phogrin independently reached the granule, although the pro-region was sorted more efficiently. Once within the regulated secretory pathway, all phogrin lumenal domain proteins were stored in functional granules for extended periods of time. Thus, phogrin possesses several domains contributing to its targeting to the secretory granule. Our findings support a model of granule biogenesis where proteins are sorted on the basis of their biochemical properties rather than via signal-dependent binding to a targeting receptor. Sorting of integral membrane proteins mediated by the lumenal domain may ensure that functionally important transmembrane molecules are included in the forming granule.     

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.     

PC1/3, PC2 and PC5/6A are targeted to dense core secretory granules by a common mechanism

There are seven members of the proprotein convertase (PC) family of secreted serine proteases that cleave their substrates at basic amino acids, thereby activating a variety of hormones, growth factors, and viruses. PC1/3, PC2 and PC5/6A are the only members of the PC family that are targeted to dense core secretory granules, where they carry out the processing of proteins that are secreted from the cell in a regulated manner. Previous studies have identified alpha-helices in the C-termini of the PC1/3 and PC2 proteases that are required for this subcellular targeting. In the current study, we demonstrate that a predicted alpha-helix in the C-terminus of PC5/6A is also critical for the ability of this domain to target a heterologous protein to the regulated secretory pathway of mouse endocrine AtT-20 cells. Analysis of the subcellular distribution of fusion proteins containing the C-terminal domains of PC1/3, PC2 and PC5/6A confirmed that all three domains have the capacity to redirect a constitutively secreted protein to the granule-containing cytoplasmic extensions. Analysis of the predicted structures formed by these three granule-sorting helices shows a correlation between their granule-sorting efficiency and the clustering of hydrophobic amino acids in their granule-targeting helices.     

AQPs and Control of Vesicle Volume in Secretory Cells

Aquaporins (AQPs) are a family of small, hydrophobic, integral membrane proteins. In mammals, they are expressed in many epithelia and endothelia and function as channels that permit water or small solutes to pass. Although the AQPs reside constitutively at the plasma membrane in most cell types, the presence of AQPs in intracellular organelles such as secretory granules and vesicles has currently been demonstrated. The secretory granules and vesicles contain secretory proteins, migrate to particular locations within the cell close to the plasma membrane and release their contents to the outside. During the process, including exocytosis, regulation of secretory granule or vesicle volume is important. This paper reviews the possible role of AQPs in secretory granules and vesicles.     

Signaling from the secretory granule to the nucleus

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40-60 nm vesicles carry conventional or small molecule neurotransmitters, and 200-400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.     

Signaling from the secretory granule to the nucleus

Neurons and endocrine cells use a complex array of signaling molecules to communicate with each other and with various targets. The majority of these signaling molecules are stored in specialized organelles awaiting release on demand: 40–60 nm vesicles carry conventional or small molecule neurotransmitters, and 200–400 nm granules contain bioactive peptides. The supply of small molecule neurotransmitters is tightly regulated by local feedback of synthetic rates and transport processes at sites of release. The larger granules that contain bioactive peptides present the secretory cell with special challenges, as the peptide precursors are inserted into the lumen of the secretory pathway in the cell soma and undergo biosynthetic processing while being transported to distant sites for eventual secretion. One solution to this dilemma in information handling has been to employ proteolytic cleavage of secretory granule membrane proteins to produce cytosolic fragments that can signal to the nucleus, affecting gene expression. The use of regulated intramembrane proteolysis to signal from secretory granules to the nucleus is compared to its much better understood role in relaying information from the endoplasmic reticulum by SREBP and ATF6 and from the plasma membrane by cadherins, Notch and ErbB4.     

Evidence for a granule targeting sequence within platelet factor 4

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed alpha-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory alpha-granule chemokine, and demonstrated that PF4 alpha-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 +/- 2% to 17 +/- 3%, p < or = 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in alpha-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNG-green fluorescent protein chimera exhibited an 82.5 +/- 1.8% colocalization with the alpha-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human alpha-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same alpha-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to alpha-granule targeting of chemokines and possibly of other alpha-granule proteins.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.     

Armed and dangerous: Toxoplasma gondii uses an arsenal of secretory proteins to infect host cells

Toxoplasma gondii is a protozoan parasite that infects a wide variety of warm-blooded animals and humans, in which it causes opportunistic disease. As an obligate intracellular parasite, T. gondii must invade a host cell to survive and replicate during infection. Recent studies suggest that T. gondii secretes a variety of proteins that appear to function during invasion or intracellular replication. These proteins originate from three distinct regulated secretory organelles called micronemes, rhoptries and dense granules. By discharging the contents of its secretory organelles at precise steps in invasion, T. gondii appears to timely deploy secretory proteins to their correct target destinations. Based on the timing of secretion and the characteristics of secretory proteins, an emerging theme is that T. gondii compartmentalizes its secretory proteins according to general function. Thus, it appears that micronemal proteins may function during parasite attachment to host cells, rhoptry proteins may facilitate parasite vacuole formation and host organellar association, and dense granule proteins likely promote intracellular replication, possibly by transporting and processing nutrients from the host cell. However, as more T. gondii secretory proteins are identified and characterized, it is likely that additional functions will be ascribed to each class of proteins secreted- by this fascinating invasive parasite.     

AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro . The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet α-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.     

Biogenesis of secretory granules

Secretory granules of neuroendocrine cells store and release peptide hormones and neuropeptides in response to various stimuli. Generation of granules from the Golgi complex involves the aggregation of cargo proteins and their sorting from non-regulated secretory molecules. Recent findings on knockout mice lacking individual granule constituents have challenged the hypothesis that an 'essential' protein for the assembly of these organelles exists, while studies on polypyrimidine tract-binding protein and ICA512/IA-2 have provided insight into the mechanisms for adjusting granule production in relation to stimulation and secretory activity.     

Sorting during transport to the surface of PC12 cells: divergence of synaptic vesicle and secretory granule proteins

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.     

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.     

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.     

The prodomain of Toxoplasma gondii GPI-anchored subtilase TgSUB1 mediates its targeting to micronemes

Subtilisin-like proteases have been proposed to play an important role for parasite survival in Toxoplasma gondii (Tg) and Plasmodium falciparum. The T. gondii subtilase TgSUB1 is located in the microneme, an apical secretory organelle whose contents mediate adhesion to the host during invasion. TgSUB1 is predicted to contain a glycosyl-phosphatidylinositol (GPI) anchor. This is unusual as Toxoplasma GPI-anchored proteins are targeted to the parasite's surface. In this study, we report that the subtilase TgSUB1 is indeed a GPI-anchored protein but contains dominant microneme targeting signals. Accurate targeting of TgSUB1 to the micronemes is dependent upon several factors including promoter strength and timing, accurate processing and folding. We analyzed the targeting domains of TgSUB1 using TgSUB1 deletion constructs and chimeras made between TgSUB1 and reporter proteins. The TgSUB1 prodomain is responsible for trafficking to the micronemes and is sufficient for targeting a reporter protein to the micronemes. Trafficking is dependent upon correct folding or other context-dependent conformation as the prodomain expressed alone is unable to reach the micromenes. Therefore, TgSUB1 is a novel example of a GPI-anchored protein in T. gondii that bypasses the GPI-dependent surface trafficking pathway to traffic to micronemes, specialized regulated secretory organelles.     

P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.     

Neutrophil tissue inhibitor of matrix metalloproteinases-1 occurs in novel vesicles that do not fuse with the phagosome

The human neutrophil granule location of precursors of matrix metalloproteinases (MMPs), MMP-8 and -9, has been established, but that of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) has not. In this study, labeling for TIMP-1, pro-MMP-8, pro-MMP-9, and established granule marker proteins reveals that TIMP-1 is mainly located in distinct oval, electron translucent organelles, a little larger than azurophil granules. A lack of labeling for the fluid phase endocytic marker, bovine serum albumin-gold, the lysosome-associated membrane protein markers, and for glycosylphosphatidylinositol-linked proteins, which are enriched in secretory vesicles, indicates the non-endosomal, non-lysosomal, and non-secretory nature of this organelle. Density gradient cofractionation with the least dense, secretory population and some pleomorphism of the organelle suggest it is a "vesicle" rather than a "granule" population. Colocalization with pro-MMP-9 or pro-MMP-8, in minor subpopulations, suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and terminal differentiation and before secretory vesicle synthesis. Pulse-chased IgG-coated latex beads and immunolabeling show that specific and azurophil granules fuse with the phagosome whereas TIMP-1 and pro-MMP-9-containing organelles do not. This suggests that these play no role in phagosomal destruction of IgG-opsonized bacteria. Separate localization and colocalization of these proteins may, however, facilitate fine regulation of extracellular proteolysis.